Under normal physiological conditions, humans and animals undergo angiogenesis, the generation of new blood vessels into a tissue or organ, in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, embryonal development, and formation of the corpus luteum, endometrium and placenta. The term “endothelium” means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels and blood vessels. The term “anti-angiogenic” or “angiogenic inhibiting activity” means the capability of a molecule to inhibit angiogenesis in general.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells are actively involved in inflammation, cell adhesion, coagulation, thrombosis, fibrinolysis, and angiogenesis. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
Persistent unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases.
During tumor growth, endothelial cells proliferate, invade the stroma, migrate toward the source of angiogenic stimuli such as cancer cells, interact with perivascular cells and stromal cells, and eventually form capillary vessels linking the tumor tissue to the circulation (J. Folkman (1995) Nat. Med. 1:27-31). Although the undoubtedly highly complex mechanism that regulates angiogenesis is yet to be understood, it is becoming clear that the initiation or termination of the process is a result of a balance between positive and negative regulators of angiogenesis. A number of angiogenic factors, often markedly upregulated in tumor tissues, have been described, including several members of the fibroblast growth factor family, such as FGF-I (G. Gimenez-Gallego et al. (1985) Science 230.:1385), FGF-2 (L. Schweigerer et al. (1987) Nature 325: 257), and those of the vascular endothelial cell growth factor family (VEGF) (D. W. Leung et al. (1989) Science 246: 1306), as well as the receptors of these growth factors (L. W. Burrus and B. B. Olwin (1989) J. Biol. Chem. 264:18647; S. Wemistrom et al. (1991) Growth Factors 4:197; B. Tennan et al (1992) Biochem. Biophys. Res. Comm. 187: 1579. C. de Vries et al., (1992) Science 255: 989). Recently, two new protein factors, proliferin and a proliferin-related protein, were found to participate in the regulation of the initiation and cessation of neovascularization in mouse placenta (Jackson D, et al. Science 266, 1581-4, 1994). All documents cited herein supra and infra are hereby expressly incorporated in their entirety by reference thereto.
Several inhibitors of angiogenesis have also been reported, including thrombospondin (D. J Good et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:6624), angiostatin (M. S. O'Reilly et al. (1994) Cell 79:315), endostatin (M. S. O'Reilly et al. (1997) Cell 88: 277) and platelet factor-4 (E. Maione et al. (1997) Science 247:77). It is apparent that normal angiogenesis is promptly activated when required, and swiftly terminated when no longer needed, whereas pathological angiogenesis, once initiated is often prolonged and difficult to stop. This indicates that the negative regulation mechanism functioning in a normal angiogenesis process is missing or suppressed in a pathological angiogenesis process. It has been suggested that proteolytic activities that release angiogenesis inhibitors from a number of precursors may account partly for down-regulation of angiogenesis, as indicated by the proteolytic activation of angiostatin from plasminogen and that of endostatin from collagen XVIII (M. S. O'Reilly, (1997) Cell 88:277). Many of the known angiogenesis regulators are pleiotrophic and can act on other cell types as well as the one that produces the regulators, although it is conceivable that endothelial cells may produce autocrine factors to suppress an angiogenic process or maintain the quiescence of a mature vasculature. It is therefore an object of the present application to describe novel autocrine negative regulators of angiogenesis of a class called Vascular Endothelial Cell Growth Inhibitors (VEGI) that are specifically expressed by endothelial cells.
Published PCT Application WO 99/23105 discloses a VEGI protein (VEGI-174) and a splice variant VEGI-251 and their corresponding nucleotide sequences, the disclosure of which is hereby expressly incorporated into the present application by reference in its entirety. Anti-angiogenic activity of N-terminal truncated forms of VEGI-174 was described. The protein VEGI-174 exhibited 20-30% sequence homology to human TNFα:, TNFβ, and the Fas ligand. A protein with a molecular weight of 22 kD was produced in an in vitro transcription and translation experiment using a cDNA clone as a template, consistent with the predicted open reading frame of 174 amino acids. This protein is herein referred to as VEGI-174. Hydrophobicity analysis of the protein predicted a 12-amino acid hydrophobic region immediately following the N-terminal segment of 14 non-hydrophobic amino acids. This was consistent with the structure of a type II transmembrane protein, similar to TNFs (B. B. Aggarwal and K. Natarajan (1996) Eur. Cytokine News. 7:93). An isoform of VEGI was also described. This protein is herein referred to as VEGI-251 which was predicted to be a membrane protein.
Recent Northern analysis of total RNA preparations from 22 different types of cultured cells of various lineages indicated that transcripts for this protein can only be detected in two lines of endothelial cells: HUVE cells and human venous endothelial cells of an early passage. A mRNA was not detected in human venous endothelial cells of a later passage, nor was it seen in human artery cells. In sharp contrast, the TNF family members are mostly expressed in immune cells (B. B. Aggarwal and K. Natarajan (1996), supra). For instance, TNFα is produced by macrophages, monocytes, neutrophils, and T cells, while TNFβ is predominantly produced by mitogen-stimulated T lymphocytes and leukocytes. Similarly, the ligands for Fas and other TNF family members, CD27, CD30, CD40, OX40, and 4-1 BB, are all expressed in cell types in the immune system. Using multiple tissue Northern blots, an EGI transcript was found to be expressed in placenta, lung, kidney, skeletal muscle, brain, liver, thymus, testis, ovary and peripheral blood lymphocytes.
Inhibition of angiogenesis in a tumor is an important approach for the treatment of cancer such as breast and other solid tumors. First of all, tumor growth and metastasis are dependent on angiogenesis. It has been shown in a model system that blocking the capillaries of the tumor neovasculature by specifically induced coagulation gives rise to the eradication of the tumor vasculature and leads to abrogation of the tumors. In addition, it has been suggested that endothelial cells are highly proliferative in tumor tissues but are mostly quiescent in normal tissues. This makes the tumor neovasculature a specific and attractive target. Furthermore, while the characteristics of cancer cells may vary greatly in different tumors, the endothelial cell population in most solid tumors is likely to be untransformed, and thus remains homogeneous. This would apply for both human and animal subjects. It may therefore be possible to develop an antiangiogenic therapeutic agent that could be applied universally for the treatment of many different cancers.
In addition to solid tumors, other important angiogenesis-driven diseases include diabetic retinopathy, Kaposi's sarcoma, psoriasis, rheumatoid arthritis. Patients who suffer from these diseases may benefit from an anti-angiogenic therapeutic approach.
The present invention identifies and describes sequences, functions, compositions, and therapeutic uses of novel isoforms of members of the VEGI family of proteins. Two new isoforms that are termed VEGI-192a, and VEGI-192b respectively, comprise a novel N-terminal sequence that substantially alters the properties of the protein with respect to its expression, secretion, and anti-angiogenic properties.
There are disclosed two new VEGI isoforms named VEGI192a and VEGI192b, both consisting of 192 amino acid residues. Both isoforms show endothelial cell-specific expression and share a C-terminal 151-residues segment with the previously described VEGI-174 and VEGI-251. The isoforms are generated from a 17 kb human gene by alternative splicing. VEGI251, the most abundant isoform, contains a putative secretion signal. VEGI protein is detected in conditioned media of endothelial cells, human sera and VEGI251-transfected mammalian cells. Subcellular localization pattern of VEGI251 is suggestive of a secretory protein. Overexpression of VEGI251 in endothelial cells causes dose-dependent cell death. VEGI251-transfected cancer cells gave rise to xenograft tumors of reduced growth rate and microvessel density compared with tumors of VEGI174 transfectants. The invention provides a view that endothelial cell-secreted VEGI can function as an autocrine inhibitor of angiogenesis and a naturally existing modulator of vascular homeostasis.
All publications cited herein are hereby incorporated by reference in their entirety.